This invention relates generally to the production of proteins in recombinant host cells. More particularly, it relates to materials and methods for the production of mature forms of proteins from heterologous precursor polypeptides using a paired basic amino acid converting enzyme (PACE), which is expressed in selected host cells.
Many eukaryotic proteins are naturally synthesized as larger precursor polypeptides, which require further specific proteolytic processing for full maturation prior to secretion. However, many of these eukaryotic proteins or precursors when synthesized in bacteria fold incorrectly or inefficiently and, consequently, exhibit low specific activities. Posttranslational proteolysis is frequently required for the synthesis of fully biologically active, mature proteins and peptides in all eukaryotes examined, including yeast [R. S. Fuller et al., Ann. Rev. Physiol., 50:345 (1988)], invertebrates [R. H. Scheller et al., Cell, 32:7 (1983)], and mammalian cells [J. Douglass et al., Ann. Rev. Biochem., 53:665 (1984); and W. S. Sossin et al., Neuron, 2, 1407 (1989)].
One of the early events in precursor protein maturation is endoproteolytic cleavage at the carboxyl side of paired basic amino acid sequences (e.g., -Lys-Arg- and -Arg-Arg-). This kind of endoproteolytic cleavage was initially inferred from the sequences of several endocrine and neuroendocrine precursor proteins and was first proposed from studies of proinsulin [D. F. Steiner et al., Science, 157:697 (1968); R. E. Chance et al., Science, 161:165 (1968)] and the ACTH/.beta.-endorphin precursor, proopiomelanocortin (POMC) [M. Chretien and C. H. Li, Can. J. Biochem., 45:1163 (1967)]. Subsequent studies have revealed a broad spectrum of precursor proteins that require endoproteolysis at pairs of basic amino acids to yield mature peptides including serum factors [A. K. Bentley et al, Cell, 45:343 (1986)], viral proteins [C. M. Rice et al., Virology, 151:1 (1986); C. M. Rice et al., Science, 229:726 (1985); J. M. McCune et al., Cell, 53:55 (1988)], growth factors [L. E. Gentry et al., Mol. Cell Biol., 8:4162 (1988); K. Sharples et al., DNA, 6:239 (1987); M. Yanagisawa et al., Nature, 332:411 (1988); and Gray et al., Nature, 303:722 (1983)] and receptors [Y. Yosimasa, Science, 240:784 (1988)). See, also, Dickerson et al, J. Biol. Chem., 265:2462 (1990); Achsletter et al, EMBO J., 4:173 (1985); and Mizuno et al, Biochem. Biophys. Res. Commun., 144:807 (1987).
Cleavage at the site of a paired basic amino acid sequence removes many propeptides which function in a variety of roles in the processing of the mature protein. In certain cases the propeptide can mediate correct folding and disulfide bond formation within the protein sequence. In other cases the presence of the propeptide appears to be involved in .gamma.-carboxylation of glutamic acid residues in vitamin K-dependent coagulation factors. .gamma.-carboxylated proteins include Factor IX and Protein C, and certain bone-specific proteins, such as bone Gla protein/osteocalcin. The propeptide can also direct intracellular targeting and regulate the coordinate synthesis of multiple mature peptides from a single precursor polypeptide.
The sequences of the propeptide domains of certain vitamin K-dependent blood coagulation proteins have been published [See, Furie et al, Cell, 53:505 (1988)] and the size of the propeptide has been established for both Factor IX and Protein C. Factor IX is a zymogen of a serine protease that is an important component of the intrinsic pathway of the blood coagulation cascade. The protein is synthesized in the liver and undergoes extensive co- and post-translational modification prior to secretion. These modifications involve endoproteolytic processing to remove the pre- and pro-peptides, glycosylation, vitamin K-dependent .gamma.-carboxylation of 12 amino-terminal glutamic acid residues and .beta.-hydroxylation of a single aspartic acid residue.
The .gamma.-carboxyglutamic acid residues confer metal binding properties on the mature Factor IX protein and may function similarly in the processing of the other vitamin K-dependent blood clotting proteins. These .gamma.-carboxyglutamic acid residues are essential for coagulant activity. The gamma-carboxyglutamate (GLA) domain of Factor IX has also been identified as a major requirement for cell binding [Derian et al, J. Biol. Chem., 264(12):6615-6618 (1989)].
With the advance of genetic engineering, many eukaryotic proteins are being produced recombinantly in selected cell lines. For example, Chinese Hamster Ovary (CHO) DUKX cell lines producing recombinant Factor IX at high antigen levels (20 .mu.g/ml/day) have been isolated. However, only 1-2% of that recombinant protein is .gamma.-carboxylated, and therefore biologically active, in the presence of vitamin K3 [Kaufman et al, J. Biol. Chem., 261(21):9622-28 (1986)]. Additionally, amino-terminal sequencing of the recombinant protein has found that 50% of the recombinant Factor IX produced by the CHO cells retain the propeptide [Derian et al, J. Biol. Chem., 264(12): 6615-18 (1989)]. Presumably, the endoproteolytic processing enzyme of the CHO cells directing this cleavage was either saturated or simply inefficient in its function.
Several activities capable of cleaving at single or paired basic residues in vitro have been proposed as candidates for authentic mammalian precursor endoproteases. See, for example, Y. P. Loh and H. Gainer, in Brain Peptides, D. T. Krieger, M. J. Brownstein, J. B. Martin, Eds. (Wiley-Interscience, New York, 1983), pp.76-116; M. Chretien, et al. in Cell Biology of the Secretory Process (Karger, Basel, Switzerland, 1983), pp.214-246; A. J. Mason, et al., Nature, 303:300 (1983); P. J. Isackson et al., J. Cell. Biochem., 33:65 (1987); I. Lindberg et al., J. Neurochem., 42:1411 (1985); J. A. Cromlish et al., J. Biol. Chem., 261:10850 (1986); K. Docherty et al., J. Biol. Chem., 259:6041 (1984); T. C. Chang and Y. P. Loh, Endocrinology, 114, 2092 (1984); B. P. Noe et al., J. Cell. Biol., 99:578 (1984); U. P. Loh, J. Biol. Chem., 261:11949 (1986); H. W. Davidson et al., Biochem. J., 246:279 (1987); P. Gluschankof et al., J. Biol. Chem., 262:9615 (1987); C. Clamigrand et al., Biochem., 26:6018 (1987); S. O. Brennan and R. J. Peach, FEBS Letters, 229:167 (1988); R. S. Fuller et al., Proc. Natl. Acad. Sci. USA, 86:1434 (1989); K. Mizuno et al., Biochem. Biophys. Res. Comm., 159:305 (1989); I. C. Bathurst et al., Science, 235:348 (1987); and G. Thomas et al., Science, 241:226 (1988)].
Despite the fact that these candidate activities and other processing enzymes have been proposed as being involved in the propeptide processing reactions, these endoproteolytic candidates have either not been fully characterized or have not been shown to be a bona fide precursor cleaving endoprotease in vivo. The purification of proprotein cleavage enzymes has been hampered by their low levels of activity in mammalian tissue and by their membrane-associated nature. Purification of these specific proteases has been complicated additionally by non-specific cleavage of the assay substrates in vitro, and by contaminating proteases such as those released from lysosomes.
The yeast enzyme Kex2, encoded by the KEX2 gene, is a membrane-bound, Ca.sup.++ -dependent serine protease which functions late in the secretory pathway of Saccharomyces cerevisiae. The enzyme cleaves the polypeptide chains of prepro-killer toxin and prepro-.alpha.-factor of that microorganism at the paired basic amino acid sequences of Lys-Arg and Arg-Arg [D. Julius et al, Cell, 37:1075 (1984); D. Julius et al, Cell, 36:309 (1984); K. Mizuno et al., Biochem. Biophys. Res. Commun., 156:246 (1988); R. S. Fuller et al., Proc. Natl. Acad. Sci. USA, 86:1434 (1989)]. Kex-2 has been considered to be a prototypic proprotein convertase.
Recently, co-expression of the yeast KEX2 gene with POMC in mammalian BSC-40 cells (a cell line which is incapable of processing this peptide precursor) reportedly resulted in the generation, by proteolytic cleavage at pairs of basic amino acids, of authentic neuroendocrine prohormone peptides, including .gamma.-LPH and .beta.-endorphin [Thomas et al, (1988), cited above]. Foster et al, Thrombosis and Haemostasis, 62:321 (1989) have reported that the yeast KEX2gene product cleaves the Protein C precursor to a two-chain form when the yeast endoprotease of the KEX2 gene and the wild-type Protein C precursor are coexpressed. However, propeptide processing and the effect of Kex2 expression have not been studied.
Two human DNA protease sequences, designated PC2 and fur, share some structural homology with each other and with the KEX2 gene sequence. PC2, a mammalian subtilisin-like protease, was identified by amplification of a human insulinoma cDNA library by the polymerase chain reaction using KEX2-derived primers. PC2, which has been implicated in the endoproteolytic processing of prohormones, shares a partial homology to the yeast Kex2 protease, especially in the putative active site domains [Smeekens et al, J. Biol. Chem., 265:2997 (1990)]. To date, however, no functional activity has been demonstrated for the PC2 clone.
The availability of the complete Kex2 gene sequence also allowed the detection of significant homology between the Kex2 protein and "furin", the product of the partially characterized human fur gene. The fur locus was initially identified by its proximity (in the immediate upstream region) to the c-fes/fps proto-oncogene [A. J. M. Roebroek et al, EMBO J., 5:2197 (1986)]. The complete nucleotide sequence of the putative coding region of the fur gene has been reported. Upon comparison, the human fur gene product has demonstrated structural homology with the subtilisin-type serine protease encoded by the KEX2 gene of the yeast S. cerevisiae [A. M. W. van den Ouweland et al, Nucl. Acids Res., 18(3):664 (1990). This published cDNA coding sequence for fur is presented in FIG. 1 (SEQ ID NO: 1]. See, also, R. S. Fuller et al, Science, 246:482 (1989). However, no evidence of the expression of fur was reported.
An expression system has been developed which utilizes baculovirus vectors to introduce heterologous genes into insect cells in culture and subsequently effects the expression of the heterologous polypeptide. This has proven successful for the recombinant expression of some proteins [see, e.g., G. Ju et al., Curr. Communic. in Mol. Biol.--Gene Transfer Vectors for Mammalian Cells, C.S.H.L. Press (1987) pps. 39-45; and A. E. Atkinson et al., Pestic. Sci., 28:215-224 (1990)].
There remains a need in the art for a method of increasing the efficiency of proteolytic processing of precursor polypeptides in recombinant host cells.